In vivo optophysiology reveals that G-protein activation triggers osmotic swelling and increased light scattering of rod photoreceptors

Abstract

The light responses of rod and cone photoreceptors have been studied electrophysiologically for decades, largely with ex vivo approaches that disrupt the photoreceptors' subretinal microenvironment. Here we report the use of optical coherence tomography (OCT) to measure light-driven signals of rod photoreceptors in vivo. Visible light stimulation over a 200-fold intensity range caused correlated rod outer segment (OS) elongation and increased light scattering in wildtype mice, but not in mice lacking the rod G-protein alpha subunit, transducin (Gα t ), revealing these responses to be triggered by phototransduction. For stimuli that photoactivated one rhodopsin per Gα t the rod OS swelling response reached a saturated elongation of 10.0 ± 2.1%, at a maximum rate of 0.11% s −1. Analyzing swelling as osmotically driven water influx, we find the H 2 O membrane permeability of the rod OS to be (2.6 ± 0.4) × 10 −5 cm·s, comparable to that of other cells lacking aquaporin expression. Application of Van't Hoff's law reveals that complete activation of phototransduction generates a potentially harmful 20% increase in OS osmotic pressure. The increased backscattering from the base of the OS is explained by a model combining cytoplasmic swelling, translocation of dissociated G-protein subunits from the disc membranes into the cytoplasm, and a relatively higher H 2 O permeability of nascent discs in the basal rod OS. Translocation of phototransduction components out of the OS may protect rods from osmotic stress, which could be especially harmful in disease conditions that affect rod OS structural integrity.osmotic stress | phototransduction | optical coherence tomography | intrinsic optical signals | photoreceptor waveguiding

On mechanisms of light-induced deformations in photoreceptors

“…the voltage-dependent tension change resulting in membrane expansion due to the late receptor potential (LRP), which begins about 4 ms after the stimulus [2], and the much larger and longer-lasting onset of the osmotic swelling [12]. The phototransduction cascade and the timing of its various stages is well studied [26], so the magnitude and dynamics of the swelling can be compared to these processes.…”

“…Phase-resolved optical coherence tomography (pOCT) recordings in the living human eye demonstrated significant optical path length (OPL) changes in the outer segments of photoreceptors in response to light stimuli. It begins with a fast, ms-scale contraction by a few tens of nm, followed by a much slower (hundreds of ms) and much larger (hundreds of nm) elongation [11], [12]. These deformations are assumed to be linked to the underlying electrical activity in the photoreceptors and have accordingly been termed the optoretinogram, analogous to the electroretinogram.…”

“…This phenomenon has been used to classify cone types in-vivo [4]. The slow expansion has been attributed to water influx due to the osmolytes produced in the phototransduction cascade [5], [12], but the mechanism of the fast contractile response remains unknown.…”

“…Driving this tension change with the known time course of the ERP (Figure 1, [2]), we can calculate the area expansion over time for this quasi-static model. To account for the slow swelling attributed to osmotic changes during phototransduction, as well as any tension change from the late receptor potential, we also include a term linearly increasing with time [12], [25]. As shown in Supplementary Figure 1, this quasi-static model provides a poor fit to OPL changes measured in the living human eye.…”

Optoretinography: optical measurements of human cone and rod photoreceptor responses to light

“…By providing information about the phase of the light scattered back from single cells, optical coherence tomography (OCT) has been shown capable of detecting elongations much smaller than its axial resolution [3], and this capability has been leveraged to measure light-evoked OS elongation in cones with digital aberration correction [4] and hardware AO [5,6]. Conventional OCT without AO has been used to observe light-evoked rod elongation in mice [7] and its converse, rod OS shortening during dark adaptation in humans [8]. Functional imaging of single human rod photoreceptors, however, has proven challenging because their small size and rapid functional response place extraordinary demands on the resolution and speed of the imaging system.…”